β3-adrenoreceptor agonists, agonist compositions and methods of making and using the same

ABSTRACT

The invention provides compounds useful as β 3 -adrenorecptor agonists and pharmaceutical compositions comprising such compounds. The invention further includes a method for stimulating, regulating, and modulating metabolism in fats of adipose tissue in mammals by administering an effective amount of a compound of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national phase applicationcorresponding to PCT/US01/10376 filed Mar. 29, 2001, which is herebyincorporated by reference herein in its entirety.

BACKGROUND

1. Technical Field

The present invention relates to the field of β₃-Adrenoreceptor agonistsand to methods of their preparation, formulation and use to stimulate,regulate and modulate metabolism of fats in adipose tissues in animals,particularly humans and other mammals. More particularly, the presentinvention relates to the field of treating obesity and overweightconditions in animals, particularly humans and other mammals andassociated effects of conditions associated with obesity and overweight,including Type II diabetes mellitus (non-insulin dependent diabetes),insulin resistance, glucose intolerance, hypothyroidism, morbid obesity,and the like.

2. Prior Art

It was long thought that obesity was a consequence of self-indulgenceand undisciplined behavior. Obesity was seen as evidence of gluttony,through a lack of will or capacity for self-discipline The overweighthave been disparaged, and thinness has been celebrated. Indeed, theperception of thinness as a major aspect of human beauty andattractiveness has become endemic in modern culture, and overweightconditions and obesity has increasingly grown to be an unacceptablecondition for social reasons.

Masked by these cultural icons are the hard medical facts: for manyindividuals, a tendency to overweight and even obesity are oftensymptoms of organic disease or disorders of the metabolism, associatedwith serious and even life-threatening conditions. In medical economicterms alone, the costs attributable to overweight and obesity arestaggeringly high.

A wide variety of approaches to the alleviation of obesity have ebbedand flowed though modern culture, ranging from a diverse collection ofdietary strategies, to drugs, to surgical interventions, to hypnosis.All have met with indifferent success at best. Some have proved to beoutright quackery. Others have proved to be effective only for theshort-term, with loss of effectiveness over time. Still others haveproved to be generally or at least partially successful so long as theregimen is sustained, but long term compliance is difficult to attainand in some cases has proved hazardous to other aspects of health andwell-being. Some surgical procedures have had some successes, but aswith any invasive procedures, there are risks. Some approaches to weightloss and control, in the extreme, lead to conditions which arethemselves pathological, such as bulimia and anorexia nervosa. Othereffects are less extreme, but still highly undesirable, such asamennorhea, vitamin and essential nutrient deficiencies, and the like.

A great deal of the difficulty in the art and practice of obesity andoverweight management has been a consequence of attention focused on thecontrol of appetite, and reducing the amount of food intake. It has longbeen the belief of many that only by the control of caloric intake is itpossible to regulate body weight and fat deposition and utilization.Since appetite is controlled and regulated in the brain, brainpharmacology and the alteration of brain chemistry has been a primaryfocus of weight regulation and control efforts. Such approaches have ledto addictions to appetite suppressants, to primary pulmonary myopathy,cardiac valve damage, and to reports of serotonin disruptions anddisorders and psychotic episodes among users. Morbities and mortalitieshave been unacceptably high.

In another aspect of technology relating to fat is the dietary emphasison limiting dietary fat intake. For those who eat meats, there isincreasing emphasis on low fat content meats in the carcasses of theanimals employed in food stocks. Much recent efforts have been devotedto the production of beef, pork, poultry and the like with reduced fatcontent. Breeding patterns are being manipulated and generic engineeringof farm animals is being directed at lowering fat content of theanimals. The techniques of fattening of animals intended for table meatproduction is highly developed, but is gradually being limited by theemphasis on limiting dietary fats and interest in leaner carcassanimals.

Only in very recent times has obesity been addressed in relation to themetabolic pathways of the body and their role and import in fat storageand usage in the body.

Recent research has elucidated some of the mechanisms of obesity andoverweight, and has revealed that much of the limitation of prior andcurrent weight-loss techniques stems from the fact that they arebiochemically and particularly metabolically unsound and incapable ofstimulating, regulating and modulating metabolism of fats in adiposetissues. Without these characteristics, it is now known, weight loss andcontrol strategies are likely to fail or to produce conditions as bad asor worse than the weight problems they are intended to alleviate.Without heroic dedication and discipline, and even fanaticism, by thesubject, most strategies are short term in their weight loss and controleffects.

Increasing efforts have been directed to biochemical research into themechanisms of fat deposition and metabolism and into stimulating,regulating and modulating metabolism of fats in adipose tissues.Considerable recent progress has been made.

Among the biochemical work of note has been the recent recognition of arole of β-Adrenoreceptor activity in the metabolism of fats. It has beenrecognized that agonists for β-Adrenoreceptors have, in some cases,produced marked weight loss in animals, particularly humans and othermammals.

More recently, the loss of weight has been identified with theβ-Adrenoreceptor sub-type, β₃-Adrenoreceptors. The specific structure ofthe β₃-Adrenoreceptor has not been characterized, but it has beendemonstrated to be a distinct cellular structure, distinguishable fromthe β₁-Adrenoreceptor and the β₂-Adrenoreceptor sites previouslyidentified.

It has been demonstrated that compounds which are significantβ₃-Adrenoreceptor agonists produce marked weight loss in animals,particularly humans and other mammals, and that the loss is sustainedwith continuation of the administration of such compounds. Thesecompounds provide potent regulation of fat metabolism. The compoundsemployed to date are also agonists for the β₁-Adrenoreceptor and theβ₂-Adrenoreceptor sites. The lack of selectivity represents unwantedside effects of such compounds, and the compounds known asβ₃-Adrenoreceptor agonists to date are not suitable candidates fortherapeutic usage because of the unwanted and dangerous side effects.

3. Problems and Needs in the Art

The existing strategies for weight and body fat regulation areinadequate. The current strategies are ineffective, unsafe, or both.Whether through diet manipulations or through drug usage, orcombinations of such strategies, there is a lack of a clear path to safeand effective regulation of body weight and body fat which is safe andeffective, which can provide significant and long lasting relief fromthe health consequences of overweight and obesity and the conditionsassociated therewith, and from the disease conditions which areaggravated by overweight and obesity.

It is clear that the art lacks and needs therapeutic agents which arehighly potent and highly selective β₃-Adrenoreceptor agonists foreffective stimulation, regulation and modulation of metabolism of fatsin adipose tissues.

It is also clear that the art lacks and needs agents which are effectiveβ₃-Adrenoreceptor agonists free of unwanted side effects, and which aresafe for stimulating, regulating and modulating metabolism of fats inadipose tissues.

It is clear that the art lacks and needs agents which are effective atregulating the body fat of animals, particularly humans and othermammals, both in the reduction of body weight in the obese and theattendant health problems and issues, and in the production of low fattable meats from domesticated animals for human consumption.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide novel compoundswhich are safe and effective β₃-Adrenoreceptor agonists.

It is another object of the present invention to provide syntheses ofsuch β₃-Adrenoreceptor agonists.

Another object of the present invention is the provision of safe andeffective β₃ Adrenoreceptor formulations for administration tostimulate, regulate and modulate metabolism of fats in adipose tissuesin animals, particularly humans and other mammals.

Still another object of the present invention is to provide safe andeffective administration of β₃-Adrenoreceptor agonists for stimulating,regulating and modulating metabolism of fats in adipose tissues inanimals, particularly humans and other mammals.

Yet another object of the present invention is to provide a safe andeffective regimen for causing and promoting weight loss in humans, andfor the maintenance of healthy and personally desired body fat levels.

Still another object of the present invention is to provide safe andeffective adjuncts to the husbandry of domesticated animals for theproduction of low fat dietary meats for human consumption.

The primary objective of the present invention is to provide for weightand body fat regulation through modalities which are effective and safe.The present invention provides a clear path to safe and effectiveregulation of body weight and body fat which is safe and effective,which can provide significant and long lasting relief from the healthconsequences of overweight and obesity and the conditions associatedtherewith, and from the disease conditions which are aggravated byoverweight and obesity.

These and related objectives are met by the terms of the presentinvention as set out in detail in the following specification anddefined in the claims appended hereto.

SUMMARY OF THE INVENTION

Compounds which are highly potent and highly specific β₃-Adrenoreceptoragonists are provided. The compounds are formulated into pharmaceuticalpreparations and administered for stimulating, regulating and modulatingmetabolism of fats in adipose tissues in animals, particularly humansand other mammals.

The compounds of the invention have the structure:

R₁ and R₂ are each independently members selected from the groupconsisting of H, OH, Cl, NO₂, CH₃SO₂NH, NH₂, CH₃O and weak acids of thestructure R₇—NH, where R₇ is an acyl group, wherein at least one of R₁and R₂ is OH. It is generally preferred that R₂ be OH.

R₃, R₄ and R₅ are variously and independently members selected from I,Br, Cl, F, OCH₃, CH₃, alkyl, alkylaryl, aminoalkyl, thioalkyl, andO-alkyl. Preferably, R4 and R5 are each a halogen, the same ordifferent.

R₆ is an acid moiety which forms an acid salt with the NH group. R₆ isdesirably HCl or (COOH)₂.

While the racemic mixtures are active, selective, and bioavailable, wehave found that the isolated isomers are ordinarily of more particularinterest. The S(−) isomers are preferred, as they will be found to havethe highest selectivity and the highest bioavailability. The R(+)isomers are also of interest, as the R-isomers are in some cases easierto isolate.

The compounds are formulated into pharmaceutical carriers to serve ashighly selective, effective and safe β₃-Adrenoreceptor agonists toprovide long term weight control.

In humans, the compositions are administered to control body fat levels,and to maintain acceptable body fat levels over time.

In domesticated animals, the compositions are administered to attaindesirably low fat content in carcass meats intended for humanconsumption.

DETAILED DESCRIPTION

In the following description of the invention, the compounds of thepresent invention, the method of their synthesis, their formulation intopharmaceutical compositions suitable for administration, and the methodof their use for stimulating, regulating and modulating metabolism offats in adipose tissues in animals, particularly humans and othermammals.

The discussion and presentation of bioactivity information and data inthe present description is made in compliance with the standards of theJournal of Medicinal Chemistry. All chemical compounds are named inaccordance with the standards of the American Chemical Society rules ofstandard nomenclature, employing accepted “trivial names” whereapplicable. All chemical structures are shown in “skeletal” form, forclarity in understanding the most significant considerations andinformation about the structures, with implicit hydrogen atoms notrelevant to the conformation of structures not shown, in the fashiontypically employed in the Journal of Medicinal Chemistry and many otherjournals of chemistry. The use of such structural notation is mostconvenient to understand the structures of such molecules, and those ofordinary levels of skill in the relevant arts are accustomed to suchrepresentations and are readily able to identify and understand such“skeletal” structures, including the implicit hydrogen atoms not shown.

Introduction

The risks and unacceptable levels of adverse consequences of many weightcontrol and weight loss strategies available to individuals and to themedical community make the development of safe and effective modalitiesfor stimulating, regulating and modulating metabolism of fats in adiposetissues an important need in the art and in society as a whole.

The importance of regulating dietary fat intake, and particularlysaturated animal fat, has long been recognized. Consumption of meats isprimary in the diet in most developed countries, and substantial effortshave been devoted to the development of leaner animals, among otherstrategies, to facilitate regulating and limiting of dietary intake ofsaturated animal fats.

In the present invention, the highly desirable goals of stimulating,regulating and modulating metabolism of fats in adipose tissues inanimals, particularly humans and other mammals through the modality ofadministering a pharmaceutical formulation of one or more compoundswhich are β₃-Adrenoreceptor selective agonists is provided.

The regulatory and modulatory effect of the compounds of the presentinvention are dependent on continued administration over time, and theattainment of an equilibrium state which is believed to be dosedependent. In that fashion, the present invention affords the control ofbody fat in animals, particularly humans and other mammals, oversustained periods, at desirable levels of body fat and/or body massindices, as defined in the medical literature.

Overview of the Invention

Safe and effective control of body fat and body mass indices have been along sought but quite elusive goal for the medical community. Themodalities in use over the past half century have proved to be bothdangerous and limited in effectiveness. The longer the effort issustained, in general, the higher the risk and the lower theeffectiveness.

The weight loss effect of β-Adrenoreceptor agonists generally has beenknown per se for a considerable period. That recognition has not led tosafe and effective weight loss or regulation because of the copious andhighly dangerous side effects.

The recent discovery of the β₃-Adrenoreceptor and its focal role in fatmetabolism holds the promise of the employment of β₃-Adrenoreceptoragonists in weight loss and regulation. Through the development ofcompounds which are highly selective for the β₃-Adrenoreceptor withoutactivation of the β₁ Adrenoreceptor and β₂ Adrenoreceptor the presentinvention makes that goal attainable.

The β₃-Adrenoreceptor has not been characterized to date, which makesthe search for safe and effective agonists with the required highselectivity a difficult and arduous task. Without a clear understandingof the receptor binding site, the design of effective compounds is basedlargely on structural activity correlations which are uncertain,unpredictable and unreliable. Even the most minor changes in structurecan produce wide deviations in binding affinity, binding specificity,and agonist activity. The compounds of the present invention attain thehigh affinity for the β₃-Adrenoreceptor, the low affinity for the β₁Adrenoreceptor and the β₂ Adrenoreceptor required for effectiveselectivity and freedom from adverse side effects, and high levels ofagonist activity to make the compounds effect in their required role infat metabolism.

THE β-ADRENORECEPTOR FAMILY

β Adrenoreceptors have long been known and have been studied for theirrole in response to the catechol amine hormones adrenaline(epinephrine), noradrenaline (norepinephrine) and dopamine.

Adrenaline, to exemplify the biochemical action of these catechol aminehormones, is a primary agonist for these receptors in the body, andactivates metabolic processes within the cells to which it binds.Adrenaline is associated with specific cellular processes which aredependent upon the nature of the cell to which it is bound. The actionof adrenaline on the cell is to activate an enzyme within the cell,adenylate cyclase. The adenylate cyclase in turn catalyses furtherreactions within the target cell, typically beginning an enzyme cascadeuntil the enzyme is broken down or deactivated by cellular regulatorymechanisms. The primary action of adenylate cyclase is the conversion ofATP to cAMP (cyclic adenosine monophosphate or “cyclic adenylate”).

In the liver cells, the cAMP activates, in turn, an enzyme cascade whichcatalyses the conversion of glycogen into glucose and inhibits theconversion of glucose into glycogen, greatly increasing extra-cellularlevels of blood glucose in the body.

In muscle tissues, cAMP triggers the breakdown of glycogen into lactateand ATP, providing high levels of ATP to support high levels of muscularactivity. In the heart muscle, in particular, the effect is hypertensiveand is accompanied by vasodilation throughout the body, increasing bloodflow and transport of blood glucose to the cells.

(β-blockers are among the commonly prescribed drugs in the field ofcardiology. For the hypertensive patient, competitive binding of theblocking agent to the β Adrenoreceptors modulates and limits theadditional hypertensive action of adrenaline on the heart muscle. Theβ-blockers may be employed in combination with vasodilators, decreasingthe resistance to blood flow peripherally without increasing the heartrate and strength of contraction. A reduction in blood pressure and thework requirement on the heart muscle results.)

In the lung, cAMP acts to cause bronchodilation which, when combinedwith increased blood flow, supplies higher levels of oxygen transport.

(Adrenaline, or epinephrine, is widely employed to stimulatebronchodilation in the treatment of asthma and allergenic reactionswhich constrict the bronchia.)

Others of the catechol amine hormones have comparable activities.

The release of free fatty acids from adipose tissue has been observed asan action provided by β Adrenoreceptor agonists.

A variety of β Adrenoreceptor agonists and blockers have been known forsome time, and have proved to be a fruitful field for drug development.

It has been recognized that there are sub-types of the β Adrenoreceptor,designate the β₁ Adrenoreceptor and the β₂ Adrenoreceptor. Lands, etal., “Differentiation of Receptor Systems Activated by SympathomimeticAmines” Nature, 214:597-598 (1967). Lands, et al., associate the releaseof free fatty acids from adipose tissue with β₁ Adrenoreceptoractivation.

Subsequent studies have provided a spectrum of β Adrenoreceptor agonistsand blockers. Among the blockers are both competitive andnon-competitive (non-equilibrium) binding agents. Some of such agentsare ubiquitous in their action, while others exhibit varying degrees ofselectivity for the two sub-types (and hence in the action responseproduced).

Selective agonist studies show both qualitative and quantitativedifferentiation of the sub-types. β₁ Adrenoreceptor activation have beendemonstrated to cause cardiac stimulation, release of free fatty acidsfrom adipose tissue, and intestinal inhibition. In contrast, β₂Adrenoreceptor activation produces broncho- and vaso-dilation.

THE β₃-ADRENORECEPTOR

Quite recently, a third sub-type of the β Adrenoreceptor family has beenidentified. Howe, R. “Beta-3 adrenergic agonists.” Drugs Future 1993,18, 529-549. It has been designated the β₃ Adrenoreceptor. It has alsobeen specifically identified with the release of free fatty acids fromadipose tissue, previously attributed by Lands et al. with the β₁Adrenoreceptor.

While β₁ Adrenoreceptor and β₂ Adrenoreceptor sites are ubiquitous, ithas been found that the β₃-Adrenoreceptor sites are more specialized andare predominantly located on adipose tissue cells, and from studies todate appear to be rather specifically associated with the metabolism offats.

β₃-ADRENORECEPTOR AGONISTS

This discovery leads quite directly to the search for selective andpotent agonists for the β₃ Adrenoreceptor for the treatment of obesityand control of weight. The search is hindered by the lack ofcharacterization of the receptor, but the information from bindingstudies and other work on β Adrenoreceptor agonists generally indicatesthat β₃ Adrenoreceptor agonists should be similar in structure to thecatechol amine hormones.

Rather little has been published to date on β₃ Adrenoreceptor agonists.See, however, Howe, R. “Beta-3 adrenergic agonists” Drugs Future 1993,18, 529-549. It is accordingly necessary to extrapolate from theinformation available about β₁ Adrenoreceptor and β₂ Adrenoreceptoragonists, and to engage in an attempt to discern structural and activityrelationships from the available data. The following comments on β₁Adrenoreceptor and β₂ Adrenoreceptor considerations summarizes what isknown in the literature upon which the effort to developβ₃-Adrenoreceptor agonists can be based.

Trimetoquinol is a potent nonspecific β-adrenoceptor (β-AR) agonistclinically used in Japan as a bronchorelaxant. Iwasawa, Y.; Kiyomoto, A.“Studies of tetrahydroisoquinolines (THI) 1. Bronchodilator activity andstructure-activity relationships.” Jap. J. Pharmacol. 1967, 17, 143-152.Optical resolution of trimetoquinol and subsequent evaluation of thestereoisomers revealed that the (S)-(−)-isomer of trimetoquinol is apotent β-adrenoceptor agonist in heart and lung tissues; whereas, the(R)-(+)-isomer acts as a selective and highly stereospecific TP receptorantagonist. Yamamoto, E.; Hirakura, M.; Sugasawa, S. “Synthesis of6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline derivatives” TetraheronSuppl. 1966, 8 (Part 1), 129-134. Mayo, J. R.; Navaran, S. S.; Akbar,H.; Miller, D. D.; Feller, D. R. “Stereodependent inhibition of humanplatelet function by the optical isomers of trimethoquinol” Biochem.Pharmacol. 1981, 30, 2237-2241. Ahn, C. H.; Romstedt, K. J.; Wallace, L.J.; Miller, D. D.; Feller, D. R. “Characterization of the inhibition ofU46619-mediated human platelet activation by the trimetoquinol isomers.Evidence for endoperoxide/thromboxane A₂ receptor blockade” BiochemPharmacol 1988, 37, 3023-33. Shin, Y.; Romstedt, K. J.; Miller, D. D.;Feller, D. R. “Stereodependent antagonism of thromboxaneA₂/prostaglandin H₂ receptor sites by trimetoquinol isomers in humanplatelets, rat vascular endothelial cells and rat vascular smooth musclecells” Pharmacol. Commun. 1993, 1, 303-312. Radioligand competitionbinding studies at β-adrenoceptor and TP receptors show highstereoselective binding (>100-fold) for the S(−)-isomer and R(+)-isomer,respectively. This stereoselectivity is also observed in the binding offluorinated trimetoquinol analogs at β-adrenoceptor. Clark, M. T.;Adejare, A.; Shams, G.; Feller, D. R.; Miller, D. D. “5-fluoro- and8-fluorotrimetoquinol: selective beta 2-adrenoceptor agonists” J MedChem 1987, 30, 86-90.

The basic catechol structure of catecholamine hormones, such asepinephrine, norepinephrine, dopamine, and the β-adrenoceptor agonistisoproterenol, is incorporated within the tetrahydroisoquinoline nucleusof trimetoquinol. In studies using mutated hamster β₂ Adrenoreceptorexpressed in Chinese hamster ovary (CHO) cells, replacement of Asp113with Asn113 abolished receptor binding of trimetoquinol and its analogs.Fraundorfer, P. F. “Functional and biochemical Characterization oftrimetoquinol (TMQ) analog interactions with β-adrenergic receptorsubtypes” Ph. D. Thesis, The Ohio State University, 1993(“Fraundorfer-2”). In addition, replacement of Ser204 and Ser207 withAla204 and Ala207 decreased the binding affinity of trimetoquinolanalogs in β₂ Adrenoreceptor to a lesser extent, but greatly diminishedtheir ability to stimulate cAMP accumulation. “Fraundorfer-2”, supra.However, both the binding and functional activities of isoproterenol aresignificantly reduced in the β₂ Adrenoreceptor Asn113, Ala204 and Ala207mutants. These results suggest that although trimetoquinol analogs mayinteract with the same amino acid residues in the binding site asisoproterenol, the contribution of catechol interactions with thesemutated β₂ Adrenoreceptors is less significant in terms of ligandbinding and may well be overshadowed by the binding contributions of thetrimethoxybenzyl group of trimetoquinol.

Substitution with fluorine or iodine on the 5- or 8-positions oftrimetoquinol resulted in only a modest (˜10-fold) increase in β₂Adrenoreceptor versus β₁ Adrenoreceptor selectivity as compared totrimetoquinol in functional and binding studies. Clark, et al., supra;Fraundorfer, P. F.; Fertel, R. H.;. Miller, D. D.; Feller, D. R.“Biochemical and pharmacological characterization of high-affinitytrimetoquinol analogs on guinea pig and human beta adrenergic receptorsubtypes: evidence for partial agonism” J Pharmacol Exp Ther 1994, 270,665-74.. In addition, it has also found that replacement of the 3′- and5′-methoxy substituent of trimetoquinol with iodine atoms (i.e., 2) iswell tolerated on both β-adrenoceptor, Fraundorfer, et al., supra, andTP receptors. Shin, Y.; Romstedt, K. J.; Miller, D. D.; Feller, D. R.“Interactions of nonprostanoid trimetoquinol analogs with thromboxaneA₂/prostaglandin H₂ receptors in human platelets, rat vascularendothelial cells and rat vascular smooth muscle cells” J Pharmacol ExpTher 1993, 267, 1017-23.; Harrold, M. W.; Gerhardt, M. A.; Romstedt, K.;Feller, D. R.; Miller, D. D. “Synthesis and platelet antiaggregatoryactivity of trimetoquinol analogs as endoperoxide/thromboxane A2antagonists” Drug Des Deliv 1987, 1, 193-207.

Interestingly, although its binding affinity at β₁ Adrenoreceptor isslightly better than trimetoquinol, compound 2 displays a much higheraffinity than trimetoquinol for β₂ Adrenoreceptor:

These earlier findings suggest that trimetoquinol analogs interact withan auxiliary site through the substituted benzyl group in addition tothe binding site shared by catecholamines. This subsite can be used toadvantage in the development of more site-selective agents. The highpotency of compound 2 seems to suggest that this auxiliary site ishydrophobic in nature. On TP receptors, the complementary binding sitesfor trimetoquinol analogs are essentially unknown. However, compound 2is a more potent TP receptor antagonist than trimetoquinol furthersuggesting that 1-benzyl ring modifications are appropriate to developagents with greater selectivity on β-Adrenoreceptor versus TP receptorsand vice versa.

The literature describes the synthesis and evaluation of iodinatedtrimetoquinol analogs designed as probes for characterizing the receptorbinding interactions, associated with the benzyl substituent oftrimetoquinol analogs and as site-selective β-adrenoceptor and TPligands. These chemical modifications provide a greater separation ofthe pharmacological activities for this class of compounds.Site-selective β-adrenoceptor agents have potential in the treatment ofcardiopulmonary diseases, non-insulin dependent diabetes (Type II) andobesity, Howe, R., “Beta-3 adrenergic agonists” Drugs Future 1993, 18,529-549, whereas highly selective TP receptor antagonists have value inthe treatment of thrombolytic disorders. Shin, supra; Shin, Y.;Romstedt, K. J.; Miller, D. D.; Feller, D. R., “Interactions ofnonprostanoid trimetoquinol analogs with thromboxane A₂/prostaglandin H₂receptors in human platelets, rat vascular endothelial cells and ratvascular smooth muscle cells” J Pharmacol Exp Ther 1993, 267, 1017-23;Shin, Y.; Romstedt, K; Doyle, K.; Harrold, M.; Gerhardt, M.; Miller, D.;Feller, D., “Pharmacologic antagonism of thromboxane A₂ receptors bytrimetoquinol analogs.” Chirality 1991, 3, 112-117.

Other known β₁ Adrenoreceptor and β₂ Adrenoreceptor agonists includeIsoproterenol, X and Y, having the structures:

While these compounds are highly active β₃-Adrenoreceptor agonists, theyare also non-selective, and also bind and activate the β₁ Adrenoreceptorand β₂ Adrenoreceptor with comparable affinities and activities. Theyare thus entirely unsuited for use in the present invention, but they doafford good basis for comparative and competitive binding studies, andare employed in the present invention for those purposes whenappropriate.

THE COMPOUNDS OF THE INVENTION

The present invention is based on the provision of β₃-Adrenoreceptoragonists in pharmaceutically acceptable carrier formulations foradministration to stimulate, regulate and modulate metabolism of fats inadipose tissues in animals, particularly humans and other mammals.

The present invention additionally provides a method for safe andeffective administration of β₃-Adrenoreceptor agonists for stimulating,regulating and modulating metabolism of fats in adipose tissues inanimals, particularly humans and other mammals.

Compounds which are highly potent and highly specific β₃-Adrenoreceptoragonists are provided. The compounds are formulated into pharmaceuticalpreparations and administered for stimulating, regulating and modulatingmetabolism of fats in adipose tissues in animals, particularly humansand other mammals.

The compounds of the invention have the structure:

R₁ and R₂ are each independently members selected from the groupconsisting of H, OH, Cl, NO₂, CH₃SO₂NH, NH₂, CH₃O and weak adds of thestructure R₇—NH, where R₇ is an acyl group, wherein at least one of R₁and R₂ is OH. It is generally preferred that R₂ be OH.

R₃, R₄ and R₅ are variously and independently members selected from I,Br, Cl, F, OCH₃, CH₃, alkyl, alkylaryl, aminoalkyl, thioalkyl, andO-alkyl. Preferably, R4 and R5 are each a halogen, the same ordifferent.

R₆ is an acid moiety which forms an acid salt with the NH group. R₆ isdesirably HCl or (COOH)₂.

While the racemic mixtures are active, selective, and bioavailable, wehave found that the isolated isomers are ordinarily of more particularinterest. The S(−) isomers are preferred, as they will be found to havethe highest selectivity and the highest bioavailability. The R(+)isomers are also effective.

The following are structures of preferred species:

It is preferred that the compounds of the present invention be furtherqualified and limited to those with high bioavailability, highselectivity and high activity for the β₃-Adrenoreceptor. In general,selectivity is highest for the S-isomers, and these are generallypreferred for these reasons. Thus, preferred species are the following:

Other species include the following:

It is particularly desirable to employ compounds of the followingstructures in the present invention, where moieties X, Y, and Z are arevariously and independently members selected from I, Br, Cl, F, OCH3,CH3, alkyl, alkylaryl, aminoalkyl, thioalkyl, and O-alkyl. Preferably, Xand Z are each a halogen, the same or different:

Preferred species of these structures having particularly goodproperties include the following compounds:

A convenient protection scheme has been devised for the synthesis of thedesired β₃-Adrenoreceptor agonists of the present invention adapted fromthe procedures disclosed in our prior U.S. application, Ser. No.09/164,047, which synthesis is hereby incorporated by reference. Asthose of ordinary skill in the art of chemical synthesis willunderstand, the procedures there are adapted to the requirements of thepresent invention by well-known and readily understood adaptations toaccommodate selection and use of differing starting reagents. The tripleprotected isoquinoline intermediates were synthesized as shown inScheme 1. The tetrahydroisoquinolines 6a-c were synthesized from theO-methyl or O-benzyl protected catecholamines 3a or 3b, respectively,and 4-nitrophenylacetic acid (4a) or 3,5-bis-trifluoromethylphenylaceticacid (4b) using methods described previously. Clark, M. T.; Adejare, A.;Shams, G.; Feller, D. R.; Miller, D. D. “5-fluoro- and8-fluorotrimetoquinol: selective beta 2-adrenoceptor agonists” J MedChem 1987, 30, 86-90.; Harrold, M. W.; Gerhardt, M. A.; Romstedt, K;Feller, D. R.; Miller, D. D. “Synthesis and platelet antiaggregatoryactivity of trimetoquinol analogs as endoperoxide/thromboxane A2antagonists” Drug Des Deliv 1987, 1, 193-207. Adejare, A.; Miller, D.D.; Fedyna, J. S.; Ahn, C. H.; Feller, D. R. “Syntheses andbeta-adrenergic agonist and antiaggregatory properties of N-substitutedtrimetoquinol analogues” J Med Chem 1986, 29, 1603-9. The amino group of6a and 6b were protected with trifluoroacetyl (TFA) andt-butyloxycarbonyl (t-BOC), respectively. The nitro groups of 7a,b werereduced via catalytic hydrogenation using Pd/C or Raney Nickel,respectively, to give the aniline derivatives 8a,b. Iodination of 8a,bwith 1 equivalent of benzyltrimethylammonium dichloroiodate (BTMACl₂I)according to Kajigaeshi et al., Kajigaeshi, S.; Kakinami, H.; Fujisaki,S.; Okamoto, T. “Halogenation using quaternary ammonium polyhalides.VII. Iodination of aromatic amines by use of benzyltrimethylammoniumdichloroiodate (I⁻)” Bull. Chem. Soc. Jpn. 1968, 61, 600-602, led to the3′-iodo analogs 9a,b. An additional 3 equivalents of BTMACl₂I added inportions over a 3 day period was required to convert 8a completely tothe diiodo derivative 10a.

Isolation of the stereo isomers is performed by known techniques,including recrystallization, column separation using HPLC, adsorptionchromotography, and the like.

What is claimed is:
 1. A compound having the structure:

wherein: one of R₁ and R₂ is OH and the other is either R₇—NH, whereinR₇ is an acyl group, or NHS(O)₂R, wherein R is alkyl or aryl, or R₁ isOH and R₂ is NO₂ or NH₂; R₃, R₄, and R₅ are each independently selectedfrom the group consisting of hydrogen, halogen, alkyl, alkylaryl,aminoalkyl, thioalkyl, O-alkyl, —NHCH₂CH₂CH₂C(O)OH, and—NHCH₂CH₂CH₂C(O)OCH₃; and R₆ is an acid moiety that forms an acid saltwith the NH group.
 2. The compound of claim 1, wherein one of R₁ and R₂is NHS(O)₂R.
 3. The compound of claim 2, wherein R is alkyl.
 4. Thecompound of claim 2, wherein R is aryl.
 5. The compound of claim 2,wherein R is tolyl.
 6. The compound of claim 2, wherein R is CH₃.
 7. Thecompound of claim 2, wherein R₄ and R₅ are each independently selectedhalogen.
 8. The compound of claim 7, wherein R₄ and R₅ are bromine. 9.The compound of claim 2, wherein R₃ is O-alkyl.
 10. The compound ofclaim 9, wherein R₃ is OCH₃.
 11. The compound of claim 1, wherein R₁ isNHS(O)₂R.
 12. The compound of claim 11, wherein R is alkyl.
 13. Thecompound of claim 11, wherein R is aryl.
 14. The compound of claim 11,wherein R is tolyl.
 15. The compound of claim 11, wherein R is CH₃. 16.The compound of claim 11, wherein R₄ and R₅ are each independentlyselected halogen.
 17. The compound of claim 16, wherein R₄ and R₅ arebromine.
 18. The compound of claim 11, wherein R₃ is O-alkyl.
 19. Thecompound of claim 18, wherein R₃ is OCH₃.
 20. The compound of claim 1,wherein R₆ is HCl or (COOH)₂.
 21. The compound of claim 1, wherein R₂ isOH, R₁ is NHS(O)₂R, R₄ and R₅ are each independently selected halogen,and R₃ is O-alkyl.
 22. The compound of claim 21, wherein R is CH₃ ortolyl.
 23. The compound of claim 21, wherein R₄ and R₅ are bromine. 24.The compound of claim 21, wherein R₃ is OCH₃.
 25. A pharmaceuticalcomposition, comprising a pharmaceutically acceptable carrier and atleast one compound having the structure:

wherein: one of R₁ and R₂ is OH and the other is either R₇—NH, whereinR₇ is an acyl group, or NHS(O)₂R, wherein R is alkyl or aryl, or R₁ isOH and R₂ is NO₂ or NH₂; R₃, R₄, and R₅ are each independently selectedfrom the group consisting of hydrogen, halogen, alkyl, alkylaryl,aminoalkyl, thioalkyl, O-alkyl, —NHCH₂CH₂CH₂C(O)OH, and—NHCH₂CH₂CH₂C(O)OCH₃; and R₆ is an acid moiety that forms an acid saltwith the NH group.
 26. The pharmaceutical composition of claim 25,wherein one of R₁ and R₂ is NHS(O)₂R.
 27. The pharmaceutical compositionof claim 26, wherein R is alkyl.
 28. The pharmaceutical composition ofclaim 26, wherein R is aryl.
 29. The pharmaceutical composition of claim26, wherein R is tolyl.
 30. The pharmaceutical composition of claim 26,wherein R is CH₃.
 31. The pharmaceutical composition of claim 26,wherein R₄ and R₅ are each independently selected halogen.
 32. Thepharmaceutical composition of claim 31, wherein R₄ and R₅ are bromine.33. The pharmaceutical composition of claim 26, wherein R₃ is O-alkyl.34. The pharmaceutical composition of claim 33, wherein R₃ is OCH₃. 35.The pharmaceutical composition of claim 25, wherein R₁ is NHS(O)₂R. 36.The pharmaceutical composition of claim 35, wherein R is alkyl.
 37. Thepharmaceutical composition of claim 35, wherein R is aryl.
 38. Thepharmaceutical composition of claim 35, wherein R is tolyl.
 39. Thepharmaceutical composition of claim 35, wherein R is CH₃.
 40. Thepharmaceutical composition of claim 35, wherein R₄ and R₅ are eachindependently selected halogen.
 41. The pharmaceutical composition ofclaim 40, wherein R₄ and R₅ are bromine.
 42. The pharmaceuticalcomposition of claim 35, wherein R₃ is O-alkyl.
 43. The pharmaceuticalcomposition of claim 42, wherein R₃ is OCH₃.
 44. The pharmaceuticalcomposition of claim 25, wherein R₆ is HCl or (COOH)₂.
 45. Thepharmaceutical composition of claim 25, wherein R₂ is OH, R₁ isNHS(O)₂R, R₄ and R₅ are each independently selected halogen, and R₃ isO-alkyl.
 46. The pharmaceutical composition of claim 45, wherein R isCH₃ or tolyl.
 47. The pharmaceutical composition of claim 45, wherein R₄and R₅ are bromine.
 48. The pharmaceutical composition of claim 45,wherein R₃ is OCH₃.
 49. A method for stimulating, regulating andmodulating metabolism of fats in adipose tissue in mammals, comprisingadministering to a mammal an effective amount of a β₃-adrenoreceptoragonist having the structure:

wherein: one of R₁ and R₂ is OH and the other is either R₇—NH, whereinR₇ is an acyl group, or NHS(O)₂R, wherein R is alkyl or aryl, or R₁ isOH and R₂ is NO₂ or NH₂; R₃, R₄, and R₅ are each independently selectedfrom the group consisting of hydrogen, halogen, alkyl, alkylaryl,aminoalkyl, thioalkyl, O-alkyl, —NHCH₂CH₂CH₂C(O)OH, and—NHCH₂CH₂CH₂C(O)OCH₃; and R₆ is an acid moiety that forms an acid saltwith the NH group.
 50. The method of claim 49, wherein one of R₁ and R₂is NHS(O)₂R.
 51. The method of claim 50, wherein R is alkyl.
 52. Themethod of claim 50, wherein R is aryl.
 53. The method of claim 50,wherein R is tolyl.
 54. The method of claim 50, wherein R is CH₃. 55.The method of claim 50, wherein R₄ and R₅ are each independentlyselected halogen.
 56. The method of claim 55, wherein R₄ and R₅ arebromine.
 57. The method of claim 50, wherein R₃ is O-alkyl.
 58. Themethod of claim 57, wherein R₃ is OCH₃.
 59. The method of claim 49,wherein R₁ is NHS(O)₂R.
 60. The method of claim 59, wherein R is alkyl.61. The method of claim 59, wherein R is aryl.
 62. The method of claim59, wherein R is tolyl.
 63. The method of claim 59, wherein R is CH₃.64. The method of claim 59, wherein R₄ and R₅ are each independentlyselected halogen.
 65. The method of claim 64, wherein R₄ and R₅ arebromine.
 66. The method of claim 59, wherein R₃ is O-alkyl.
 67. Themethod of claim 66, wherein R₃ is OCH₃.
 68. The method of claim 49,wherein R₆ is HCl or (COOH)₂.
 69. The method of claim 49, wherein R₂ isOH, R₁ is NHS(O)₂R, R₄ and R₅ are each independently selected halogen,and R₃ is O-alkyl.
 70. The method of claim 69, wherein R is CH₃ ortolyl.
 71. The method of claim 69, wherein R₄ and R₅ are bromine. 72.The method of claim 69, wherein R₃ is OCH₃.
 73. The method of claim 49,wherein the mammal is a human.